Immobilized lipase taking graft modified amino resin as carrier and preparation method of immobilized lipase

Abstract

The invention relates to the field of immobilized enzymes, in particular to immobilized lipase with graft-modified amino resin as a carrier and a preparation method thereof.The method comprises the steps that firstly, the amino resin is modified through the oxidative auto-polymerization characteristic of tannic acid, and first modified amino resin is obtained; and modifying the first modified amino resin by using fatty amine as a hydrophobic modifier to obtain second modified amino resin. Then lipase is fixed on the second modified amino resin through physical adsorption, the lipase is subjected to interfacial activation on an amphiphilic interface on the second modified amino resin, and a part of lipase is covalently bound with quinonyl in an activated state, so that the immobilized lipase with high activity and stability is obtained. The method is easy to operate, green and non-toxic, the prepared immobilized lipase is good in enzyme activity and stability, and the method can be used for preparing the immobilized lipase on a large scale, can be applied to the field of food and has industrial application potential.

Description

Technical Field

[0001] The present invention relates to the field of immobilized enzymes, and particularly to an immobilized lipase using a graft-modified amino resin as a carrier and a preparation method thereof. Background Art

[0002] Lipase (E.C.3.1.1.3), as a green and efficient biocatalyst, has a wide range of applications in the fields of biodiesel production, chiral drug preparation, and functional oil preparation. However, free lipase is very sensitive to the use environment and is easily inactivated by factors such as temperature, acidity and alkalinity, and organic solvents. Moreover, it is difficult to recycle, which severely limits its industrial application. The enzyme immobilization technology immobilizes biocatalysts on carriers through physical adsorption, covalent bonding, crosslinking, and embedding, which can significantly improve the stability and reusability of enzymes. As a typical interfacial activation enzyme, the active center of lipase is located in a hydrophobic "pocket", and the pocket is covered by a "lid" structure. The outside of the lid is hydrophilic and the inside is hydrophobic. When lipase is located at the amphiphilic interface, the conformation of lipase changes, the "lid" structure opens, and the active center is exposed. Therefore, the activity of lipase is regulated by the hydrophilic / hydrophobic microenvironment of the carrier. A suitable hydrophilic / hydrophobic microenvironment can improve the interaction between the carrier and lipase and enhance the enzyme activity and stability.

[0003] In order to regulate the hydrophilic / hydrophobic microenvironment of the carrier and improve the activity and stability of immobilized lipase, researchers have successively developed various hydrophobic modification methods for immobilized lipase carriers, mainly including the following: CN 106701729 A discloses an immobilized enzyme using a polypeptide-modified amino resin as a carrier and a preparation method thereof. Hydrophobic amino acids are grafted onto the amino resin by a chemical method to form hydrophobic polypeptide fragments with different chain lengths, forming a stable hydrophobic environment on the surface of the carrier. A functional carrier with a relatively hydrophobic surface environment is obtained, and lipase is immobilized by physical adsorption. The immobilized enzymes on the carrier modified by amino acids, amino resin-leucine residue (length 10)-lipase and amino resin-phenylalanine residue (length 10)-lipase, the residual enzyme activities are 92.75% and 80.86% respectively after storage for 12 days, and the residual enzyme activities can still reach 36.66% and 50.66% of the initial value after 21 days, showing good stability. However, this method introduces toxic chemical condensation reagents during the carrier modification process and is difficult to apply to the food industry.

[0004] CN 116463327 A discloses an immobilized lipase with an acyl chloride-modified amino resin as a carrier, its preparation method and application. The amino resin is hydrophobically modified with alkyl acyl chlorides of different carbon chain lengths, and lipase is physically adsorbed on the obtained acyl chloride-modified amino resin, and then crosslinked with the natural crosslinking agent genipin. The enzyme activity recovery rate of the obtained immobilized lipase reaches 176%, which is nearly 9 times that of the lipase immobilized by adsorption on the unmodified resin. However, this method introduces toxic alkyl acyl chlorides during the carrier modification process and is difficult to apply to the food industry.

[0005] CN 119391688 A discloses a highly stable immobilized Candida antarctica lipase B, its preparation method and application. A macroporous adsorption resin ECR1030 is hydrophobically modified with a silane coupling agent to obtain a silylated resin, and then the silylated resin is aldehyde-functionalized with dialdehyde cellulose to obtain an alkyl-aldehyde modified resin for immobilizing Candida antarctica lipase B. After the Candida antarctica lipase B undergoes interfacial activation on the surface of the alkyl-aldehyde modified resin, it covalently binds to the aldehyde groups on the surface of the alkyl-aldehyde modified resin. The prepared immobilized Candida antarctica lipase B can be reused for 50 batches with almost no loss of any activity. However, this method requires a relatively high temperature (80 °C - 95 °C) during the carrier modification process, resulting in high energy consumption and a complex process.

[0006] CN 118755788 A discloses a method for synthesizing phytosterol esters by using a bifunctional group-modified macroporous resin-immobilized lipase. The amino resin is hydrophobically modified with stearic acid using a chemical coupling agent. Glutaraldehyde is used to activate the remaining amino groups on the surface, and the immobilization of Candida rugosa lipase is achieved by the Schiff base reaction with Candida rugosa lipase and the hydrophobic interaction between the C18 alkyl group and the enzyme. The immobilized enzyme is applied to the esterification reaction of phytosterol and fatty acid. The reaction time is 48 hours, and the yield of phytosterol ester reaches as high as 95.7%. The immobilized enzyme has high catalytic activity and good reusability. After the enzyme undergoes 6 consecutive cyclic reactions, the enzyme activity can still maintain 76.2% of the initial activity. However, this method introduces highly toxic chemical coupling agents and glutaraldehyde during the carrier modification process and is difficult to apply to the food industry. Summary of the Invention

[0007] The purpose of the present invention is to provide a green, simple and low-cost method for hydrophobically modifying amino resin, precisely regulating the hydrophilic / hydrophobic microenvironment of the carrier to improve the activity and stability of immobilized lipase.

[0008] To achieve the above purpose, the present invention is realized according to the following technical solutions: On the one hand, the present invention provides a method for preparing immobilized lipase using a graft-modified amino resin as a carrier, comprising the following steps: Step S1: Dissolve tannic acid in alkaline buffer A, add amino resin, and carry out an oxidation reaction at room temperature. After filtration and drying, obtain the first modified amino resin; Step S2: Add fatty amine and the first modified amino resin into an ethanol solution respectively, carry out a grafting reaction, and after filtration and drying, obtain the second modified amino resin; Step S3: Add the second modified amino resin into the lipase solution. After adsorption, filter out the liquid, wash it with buffer B, and obtain immobilized lipase after drying.

[0009] In the above technical solution, the concentration of tannic acid dissolved in buffer A in step S1 is 1 mg / mL - 5 mg / mL. Preferably, the concentration of tannic acid dissolved in buffer A is 2 mg / mL.

[0010] In the above technical solution, buffer A in step S1 is sodium bicarbonate buffer or Tris - hydrochloric acid buffer or HEPES buffer. Preferably, buffer A is sodium bicarbonate buffer; the pH of buffer A is 8 - 9. Preferably, the pH of buffer A is 8.5.

[0011] In the above technical solution, the oxidation reaction time in step S1 is 6 - 24 h. Preferably, the oxidation reaction time is 12 h.

[0012] In the above technical solution, the model of the amino resin in step S1 is one of LX - 1000HAA, LX - 1000EPN, LX - 1000EPHA, LX - 1000NH, and LX - 1000EA.

[0013] In the above technical solution, the fatty amine in step S2 includes one or more of hexylamine, octylamine, decylamine, dodecylamine, tetradecylamine, hexadecylamine, and octadecylamine. Preferably, the fatty amine is octadecylamine.

[0014] In the above technical solution, the addition amount of the fatty amine in step S2 is 0.2 mmol - 1 mmol; preferably, the addition amount of the fatty amine is 0.4 mmol; the concentration of the ethanol solution is 50% - 100%; preferably, the concentration of the ethanol solution is 70%; the grafting reaction temperature is 40 °C - 60 °C. Preferably, the grafting reaction temperature is 50 °C; the grafting reaction time is 4 - 8 h. Preferably, the grafting reaction time is 6 h.

[0015] In the above technical solution, the lipase solution in step S3 is obtained by dissolving lipase in buffer B. Preferably, buffer B is a phosphate solution.

[0016] In the above technical solution, the mass-volume ratio of the second modified amino resin to the lipase solution in step S3 is 1 g∶5-25 mL. Preferably, the mass-volume ratio of the second modified amino resin to the lipase solution is 1 g∶10 mL.

[0017] In the above technical solution, the pH of the lipase solution in step S3 is 5-10. Preferably, the pH is 7; the adsorption time is 2-10 h. Preferably, the adsorption time is 8 h; the adsorption temperature is 25-45 °C. Preferably, the adsorption temperature is 35 °C.

[0018] In the above technical solution, the lipase is Rhizomucor miehei lipase.

[0019] On the other hand, the present invention provides an immobilized lipase prepared using a graft-modified amino resin as a carrier, which is prepared according to any of the above methods.

[0020] Compared with the prior art, the present invention has the following beneficial effects: By precisely designing the hydrophilic / hydrophobic microenvironment of the immobilized lipase carrier, the activity and stability of the immobilized lipase are improved. First, a layer of oxidized tannic acid coating is formed on the amino resin by utilizing the property of tannic acid to oxidize and self-polymerize in an alkaline environment. Different amounts and carbon chain lengths of fatty amines are anchored on the surface of the tannic acid coating through Schiff base and Michael addition reactions between the quinone active groups on the oxidized tannic acid coating and the amino groups of fatty amines for hydrophobic modification, obtaining a second modified amino resin. Subsequently, the lipase is physically adsorbed on the second modified amino resin, and interfacial activation occurs at the amphiphilic interface of the lipase on the second modified amino resin. A part of the lipase covalently binds to the quinone group in the activated state, thereby obtaining an immobilized lipase with high activity and stability. In the modification process of the amino resin of the present invention, only an ethanol solution is used, which is a safe and environmentally friendly method for preparing immobilized lipase. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 It is a technical route diagram of the method for preparing an immobilized lipase using a graft-modified amino resin as a carrier according to the present invention.

[0023] Figure 2 It is a schematic diagram of the graft-modified amino resin according to the present invention.

[0024] Figure 3 Effect of enzyme dosage on enzyme activity and immobilization efficiency.

[0025] Figure 4 Effect of pH of enzyme solution on enzyme activity and immobilization efficiency.

[0026] Figure 5 Effect of adsorption time on enzyme activity and immobilization efficiency.

[0027] Figure 6 Effect of adsorption temperature on enzyme activity and immobilization efficiency.

[0028] Figure 7 Characterization of thermal stability of immobilized lipase.

[0029] Figure 8 Characterization of storage stability of immobilized lipase. Specific implementation mode

[0030] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments.

[0031] As Figure 1 shown, the present invention proposes a method for preparing immobilized lipase using graft-modified amino resin as a carrier, including the following steps: Step S1: Dissolve tannic acid in alkaline buffer A, add amino resin, and stir at room temperature for an oxidation reaction. After filtration and drying, obtain the first modified amino resin; Step S2: Add fatty amine and the first modified amino resin into an ethanol solution respectively, carry out a graft reaction, and after filtration and drying, obtain the second modified amino resin; Step S3: Add the second modified amino resin into the lipase solution. After adsorption, filter out the liquid, wash it with buffer B, and obtain immobilized lipase after drying.

[0032] Combined with Figure 2, the principle of the method for preparing the immobilized lipase of the present invention is as follows: As a typical interfacial activation enzyme, lipase requires a suitable hydrophilic / hydrophobic microenvironment to activate lipase at the interface. However, amino resin exhibits hydrophilicity due to the rich amino groups on its surface. Directly used as an immobilized enzyme carrier, it cannot effectively activate lipase, resulting in low enzyme activity. Therefore, it is necessary to hydrophobically modify the amino resin to obtain a suitable hydrophilic / hydrophobic microenvironment and improve enzyme activity and stability. In step S1, tannic acid self-polymerizes to form an oxidized tannic acid coating under alkaline conditions. At the same time, since its phenolic hydroxyl groups are easily oxidized to form quinones in an alkaline environment, the quinone groups, as active groups, can react with the amino groups on the amino resin through Schiff base and Michael addition reactions to form covalent bonds, thereby helping tannic acid to quickly and tightly form an oxidized tannic acid coating on the amino resin. In step S2, hydrophobic fatty amines with different numbers and carbon chain lengths are anchored on the surface of the tannic acid coating by the Schiff base and Michael addition reactions between the remaining quinone active groups on the oxidized tannic acid coating and the amino groups of fatty amines to obtain immobilized enzyme carriers with different hydrophilic / hydrophobic properties. In step S3, lipase is physically adsorbed on the second modified amino resin, and lipase undergoes interfacial activation at the amphiphilic interface on the second modified amino resin. Part of the lipase covalently binds to the quinone groups in the activated state, thereby obtaining an immobilized lipase with high activity and stability; The present invention innovatively introduces an oxidized tannic acid coating as a multifunctional platform, hydrophobically modifies and immobilizes enzymes through Schiff base and Michael addition reactions, without introducing additional cross-linking agents. By controlling the chain length and grafting density of fatty amines, an amphiphilic interface with adjustable hydrophilic / hydrophobic properties is constructed on the amino resin, providing a suitable immobilization microenvironment for lipase and improving the activity and stability of the immobilized lipase.

[0033] The method for detecting enzyme activity in the following examples is as follows: Definition of enzyme activity: Under certain reaction conditions, the amount of enzyme required for the immobilized enzyme to catalyze the esterification of lauric acid and n-propanol to produce 1 μmol of propyl laurate per minute is defined as one enzyme activity unit (U).

[0034] Determination method: Premix 80.1 g of lauric acid, 24 g of n-propanol and 3.2 g of distilled water evenly. Take 5.36 g of the premixed substrate and place it in a 50 mL ground glass stoppered conical flask. Place it in a constant temperature water bath shaker with a rotation speed of 200 rpm and a temperature of 60 °C and oscillate and preheat for 10 min. Then add about 15 mg of immobilized lipase. After reacting for 20 min, immediately take 30 μL of the reaction product and dissolve it in 970 μL of n-heptane. Use gas chromatography (GC) to determine the contents of lauric acid and propyl laurate. Use the following formula to calculate the enzyme activity:

[0035] Where N is the amount of lauric acid in the substrate (mol); C is the ratio of lauric acid converted to propyl laurate; W is the mass of the enzyme (g); and t is the reaction time (min).

[0036] The fixed efficiency is calculated as follows:

[0037] Where C0 is the protein content of the enzyme solution before immobilization, and C1 is the protein content of the enzyme solution after immobilization.

[0038] The main raw materials and reagents in the examples are as follows: tannic acid: purity 98%, Maclean's reagent; octadecylamine: purity 99%, Maclean's reagent; hexadecylamine: purity 95%, Maclean's reagent; tetradecylamine: purity 96%, Maclean's reagent; dodecylamine: purity 98%, Maclean's reagent; decylamine: purity 98%, Maclean's reagent; octylamine: purity 99%, Maclean's reagent; hexylamine: purity 99%, Maclean's reagent; Rhizomucor miehei lipase: enzyme activity 1007 U / mL, Qingdao Weilan Biotechnology Co., Ltd.

[0039] Buffer A was prepared by weighing 50 mmol of one of sodium bicarbonate, Tris (tris(hydroxymethylaminomethane), and HEPES (hydroxyethylpiperazineethanesulfonic acid), dissolving it in 800 mL of distilled water, adjusting the pH to the corresponding value with sodium hydroxide solution, and then diluting the volume to 1 L.

[0040] Buffer B was prepared as follows: 50 mmol of potassium dihydrogen phosphate and dipotassium hydrogen phosphate were weighed and dissolved in 800 mL of distilled water. The pH value was adjusted to the corresponding value with sodium hydroxide solution, and the volume was made up to 1 L. Example 1

[0041] Weigh 10 g of wet LX-1000 HAA amino resin into 200 mL of sodium bicarbonate buffer solution (pH 8.5, 50 mM), add 0.4 g of tannic acid (the concentration of tannic acid dissolved in buffer A is 2 mg / mL), stir at room temperature for 12 h, filter and then dry under vacuum to obtain the first modified amino resin. Add 1 g of the first modified amino resin into 25 mL of ethanol solution (70%, v / v), and respectively add 0.4 mmol of octadecylamine, hexadecylamine, tetradecylamine, dodecylamine, decylamine, octylamine, and hexylamine, react at 50 °C for 6 h, filter and dry under vacuum to obtain the second modified amino resin. Dissolve 2.5 g of Rhizomucor miehei lipase in 50 mL of phosphate buffer solution (pH 7, 50 mM), centrifuge and take the supernatant to obtain the lipase solution. Take 4 mL of the above lipase solution, add 0.2 g of the second modified amino resin, immobilize at 30 °C for 10 h, filter and separate the immobilized lipase, wash the immobilized lipase with phosphate buffer solution until there is no protein residue in the filtrate, and vacuum dry the washed immobilized product at 40 °C for 4 h.

[0042] Compare and analyze the effects of the types of fatty amines on the immobilization efficiency and enzyme activity, and determine the optimal type of fatty amine. As can be seen from Table 1, with the increase of the carbon chain length of the fatty amine, the enzyme activity shows an increasing trend. Therefore, using octadecylamine as the hydrophobic modifier has the best effect on improving the enzyme activity, and the highest enzyme activity can reach 10823.54 U / g, and the immobilization efficiency is 52.35% at this time.

[0043] Example 2

[0044] Weigh 10 g of wet LX-1000HAA amino resin into 200 mL of sodium bicarbonate buffer (pH 8.5, 50 mM), add 0.4 g of tannic acid (the concentration of tannic acid dissolved in buffer A is 2 mg / mL), stir at room temperature for 12 h, filter and then dry in vacuum to obtain the first modified amino resin. Add 1 g of the first modified amino resin into 25 mL of ethanol solution (70%, v / v), and add 0.2 mmol, 0.4 mmol, 0.6 mmol, 0.8 mmol, 1.0 mmol of octadecylamine respectively, react at 50 °C for 6 h, filter and dry in vacuum to obtain the second modified amino resin. Dissolve 2.5 g of Rhizomucor miehei lipase in 50 mL of phosphate buffer (pH 7, 50 mM), take the supernatant after centrifugation to obtain the lipase solution. Take 4 mL of the above lipase solution, add 0.2 g of the second modified amino resin, immobilize at 30 °C for 10 h, filter and separate the immobilized lipase, wash the immobilized lipase with phosphate buffer until there is no protein residue in the filtrate, and vacuum dry the washed immobilized product at 40 °C for 4 h.

[0045] Compare and analyze the effect of the addition amount of octadecylamine on the immobilization efficiency and enzyme activity, and determine the optimal addition amount of octadecylamine. As can be seen from Table 2, as the addition amount of octadecylamine increases, the enzyme activity shows a trend of increasing first and then decreasing. When the addition amount of octadecylamine is 0.4 mmol, it has the highest immobilization efficiency and enzyme activity, and the immobilization efficiency and enzyme activity can reach 51.51% and 10781.58 U / g respectively.

[0046] Example 3

[0047] Weigh 10 g of wet LX-1000HAA amino resin into 200 mL of sodium bicarbonate buffer solution (pH 8.5, 50 mM), add 0.4 g of tannic acid (the concentration of tannic acid dissolved in buffer solution A is 2 mg / mL), stir at room temperature for 12 h, filter and then dry in vacuum to obtain the first modified amino resin. Add 1 g of the first modified amino resin into 25 mL of ethanol solution (70%, v / v), add 0.4 mmol of octadecylamine, react at 50 °C for 6 h, filter and dry in vacuum to obtain the second modified amino resin. Dissolve 2.5 g of Rhizomucor miehei lipase in 50 mL of phosphate buffer solution (pH 7, 50 mM), centrifuge and take the supernatant to obtain the lipase solution. Take 1 mL, 2 mL, 3 mL, 4 mL, 5 mL of the above lipase solution respectively, add 0.2 g of the second modified amino resin, fix at 30 °C for 8 h, filter and separate the immobilized lipase, wash the immobilized lipase with phosphate buffer solution until there is no protein residue in the filtrate, and vacuum dry the washed immobilized product at 40 °C for 4 h.

[0048] Compare and analyze the influence of enzyme addition amount on the immobilization efficiency and enzyme activity, and determine the optimal enzyme addition amount. As Figure 3 shown, with the increase of the enzyme addition amount, the enzyme activity shows a trend of first increasing and then decreasing. When the enzyme addition amount is 10 mL / g, the enzyme activity is the highest, and the enzyme activity can reach 10909.62 U / g. At this time, the immobilization efficiency is 69.83%. Example 4

[0049] Weigh 10 g of wet LX-1000HAA amino resin into 200 mL of sodium bicarbonate buffer solution (pH 8.5, 50 mM), add 0.4 g of tannic acid (the concentration of tannic acid dissolved in buffer solution A is 2 mg / mL), stir at room temperature for 12 h, filter and then dry in vacuum to obtain the first modified amino resin. Add 1 g of the first modified amino resin into 25 mL of ethanol solution (70%, v / v), add 0.4 mmol of octadecylamine, react at 50 °C for 6 h, filter and dry in vacuum to obtain the second modified amino resin. Dissolve 2.5 g of Rhizomucor miehei lipase in 50 mL of phosphate buffer solutions with pH values of 5, 6, 7, 8, 9, 10 (50 mM) respectively, centrifuge and take the supernatant to obtain the lipase solution. Take 2 mL of the above lipase solution, add 0.2 g of the second modified amino resin, fix at 30 °C for 10 h, filter and separate the immobilized lipase, wash the immobilized lipase with phosphate buffer solution until there is no protein residue in the filtrate, and vacuum dry the washed immobilized product at 40 °C for 4 h.

[0050] Compare and analyze the effects of the pH of the enzyme solution on the immobilization efficiency and enzyme activity, and determine the optimal enzyme addition amount. As Figure 4 shown, as the pH of the enzyme solution increases, the enzyme activity shows a trend of first increasing and then decreasing. When the pH of the enzyme solution is 7, the enzyme activity is the highest, and the enzyme activity can reach 11002.84 U / g. At this time, the immobilization efficiency is 68.12%. Example 5

[0051] Weigh 10 g of wet LX-1000HAA amino resin into 200 mL of sodium bicarbonate buffer solution (pH 8.5, 50 mM), add 0.4 g of tannic acid (the concentration of tannic acid dissolved in buffer A is 2 mg / mL), stir at room temperature for 12 h, filter and vacuum dry to obtain the first modified amino resin. Add 1 g of the first modified amino resin into 25 mL of ethanol solution (70%, v / v), add 0.4 mmol of octadecylamine, react at 50 °C for 6 h, filter and vacuum dry to obtain the second modified amino resin. Dissolve 2.5 g of Rhizomucor miehei lipase in 50 mL of phosphate buffer solution (pH 7, 50 mM), centrifuge and take the supernatant to obtain the lipase solution. Take 2 mL of the above lipase solution, add 0.2 g of the second modified amino resin, and immobilize at 30 °C for 2, 4, 6, 8, and 10 h respectively. Filter and separate the immobilized lipase, wash the immobilized lipase with phosphate buffer solution until there is no protein residue in the filtrate, and vacuum dry the washed immobilized product at 40 °C for 4 h.

[0052] Compare and analyze the effects of the immobilization time of the enzyme solution on the immobilization efficiency and enzyme activity, and determine the optimal immobilization time. As Figure 5 shown, as the immobilization time increases, the enzyme activity and immobilization efficiency show an increasing trend and then gradually reach equilibrium. When the immobilization time of the enzyme solution is 8 h, the enzyme activity is the highest, and the enzyme activity can reach 11363.91 U / g. At this time, the immobilization efficiency is 64.32%. Example 6

[0053] Weigh 10 g of wet LX-1000HAA amino resin into 200 mL of sodium bicarbonate buffer (pH 8.5, 50 mM), add 0.4 g of tannic acid (the concentration of tannic acid dissolved in buffer A is 2 mg / mL), stir at room temperature for 12 h, filter and then dry in vacuum to obtain the first modified amino resin. Add 1 g of the first modified amino resin into 25 mL of ethanol solution (70%, v / v), add 0.4 mmol of octadecylamine, react at 50 °C for 6 h, filter and dry in vacuum to obtain the second modified amino resin. Dissolve 2.5 g of Rhizomucor miehei lipase in 50 mL of phosphate buffer (pH 7, 50 mM), centrifuge and take the supernatant to obtain the lipase solution. Take 2 mL of the above lipase solution respectively, add 0.2 g of the second modified amino resin, fix at 25 °C, 30 °C, 35 °C, 40 °C, 45 °C for 8 h respectively, filter and separate the immobilized lipase, wash the immobilized lipase with phosphate buffer until there is no protein residue in the filtrate, and vacuum dry the washed immobilized product at 40 °C for 4 h.

[0054] Compare and analyze the effects of the immobilization temperature of the enzyme solution on the immobilization efficiency and enzyme activity, and determine the optimal immobilization temperature. As Figure 6 shown, with the increase of the immobilization temperature, the enzyme activity and immobilization efficiency show a trend of increasing first and then decreasing. When the immobilization temperature of the enzyme solution is 35 °C, the enzyme activity is the highest, and the enzyme activity can reach 11638.53 U / g. At this time, the immobilization efficiency is 68.58%. Example 7

[0055] Weigh 10 g of wet LX-1000EPN amino resin into 200 mL of sodium bicarbonate buffer (pH 8.5, 50 mM), add 0.2 g of tannic acid (the concentration of tannic acid dissolved in buffer A is 1 mg / mL), stir at room temperature for 24 h, filter and then dry in vacuum to obtain the first modified amino resin. Add 1 g of the first modified amino resin into 25 mL of ethanol solution (70%, v / v), add 0.4 mmol of octadecylamine, react at 50 °C for 6 h, filter and dry in vacuum to obtain the second modified amino resin. Dissolve 2.5 g of Rhizomucor miehei lipase in 50 mL of phosphate buffer (pH 7, 50 mM), centrifuge and take the supernatant to obtain the lipase solution. Take 2 mL of the above lipase solution, add 0.2 g of the second modified amino resin, fix at 35 °C for 8 h, filter and separate the immobilized lipase, wash the immobilized lipase with phosphate buffer until there is no protein residue in the filtrate, and vacuum dry the washed immobilized product at 40 °C for 4 h.

[0056] The properties of the obtained immobilized lipase were characterized, and the results showed that the immobilization efficiency was 65.52% and the enzyme activity was 10325.32 U / g. Example 8

[0057] Weigh 10 g of wet LX-1000HAA amino resin into 200 mL of Tris-HCl buffer (pH 8, 50 mM), add 0.4 g of tannic acid (the concentration of tannic acid dissolved in buffer A is 2 mg / mL), stir at room temperature for 18 h, filter and then dry in vacuum to obtain the first modified amino resin. Add 1 g of the first modified amino resin into 25 mL of ethanol solution (90%, v / v), add 0.4 mmol of octadecylamine, react at 40 °C for 8 h, filter and dry in vacuum to obtain the second modified amino resin. Dissolve 2.5 g of Rhizomucor miehei lipase in 50 mL of phosphate buffer (pH 7, 50 mM), centrifuge and take the supernatant to obtain the lipase solution. Take 2 mL of the above lipase solution, add 0.2 g of the second modified amino resin, fix at 35 °C for 8 h respectively, filter to separate the immobilized lipase, wash the immobilized lipase with phosphate buffer until there is no protein residue in the filtrate, and vacuum dry the washed immobilized product at 40 °C for 4 h.

[0058] The properties of the obtained immobilized lipase were characterized, and the results showed that the immobilization efficiency was 62.43% and the enzyme activity was 9823.73 U / g. Example 9

[0059] Weigh 10 g of wet LX-1000NH amino resin into 200 mL of HEPES buffer (pH 9, 50 mM), add 0.6 g of tannic acid (the concentration of tannic acid dissolved in buffer A is 3 mg / mL), stir at room temperature for 10 h, filter and then dry in vacuum to obtain the first modified amino resin. Add 1 g of the first modified amino resin into 25 mL of ethanol solution (50%, v / v), add 0.4 mmol of octadecylamine, react at 60 °C for 4 h, filter and dry in vacuum to obtain the second modified amino resin. Dissolve 2.5 g of Rhizomucor miehei lipase in 50 mL of phosphate buffer (pH 7, 50 mM), centrifuge and take the supernatant to obtain the lipase solution. Take 2 mL of the above lipase solution, add 0.2 g of the second modified amino resin, fix at 35 °C for 8 h respectively, filter to separate the immobilized lipase, wash the immobilized lipase with phosphate buffer until there is no protein residue in the filtrate, and vacuum dry the washed immobilized product at 40 °C for 4 h.

[0060] The properties of the obtained immobilized lipase were characterized, and the results showed that the immobilization efficiency was 64.23% and the enzyme activity was 9547.68 U / g. Example 10

[0061] Weigh 10 g of wet LX-1000EA amino resin into 200 mL of HEPES buffer (pH 8.5, 50 mM), add 0.8 g of tannic acid (the concentration of tannic acid dissolved in buffer A is 4 mg / mL), stir at room temperature for 8 h, filter and then dry in vacuum to obtain the first modified amino resin. Add 1 g of the first modified amino resin into 25 mL of ethanol solution (80%, v / v), add 0.4 mmol of octadecylamine, react at 45 °C for 7 h, filter and dry in vacuum to obtain the second modified amino resin. Dissolve 2.5 g of Rhizomucor miehei lipase in 50 mL of phosphate buffer (pH 7, 50 mM), centrifuge and take the supernatant to obtain the lipase solution. Take 2 mL of the above lipase solution, add 0.2 g of the second modified amino resin, immobilize at 35 °C for 8 h respectively, filter to separate the immobilized lipase, wash the immobilized lipase with phosphate buffer until there is no protein residue in the filtrate, and vacuum dry the washed immobilized product at 40 °C for 4 h.

[0062] The properties of the obtained immobilized lipase were characterized, and the results showed that the immobilization efficiency was 64.57% and the enzyme activity was 10435.97 U / g. Example 11

[0063] Weigh 10 g of wet LX-1000EPHA amino resin into 200 mL of Tris-HCl buffer (pH 8.5, 50 mM), add 1 g of tannic acid (the concentration of tannic acid dissolved in buffer A is 5 mg / mL), stir at room temperature for 6 h, filter and then dry in vacuum to obtain the first modified amino resin. Add 1 g of the first modified amino resin into 25 mL of ethanol solution (100%, v / v), add 0.4 mmol of octadecylamine, react at 55 °C for 5 h, filter and dry in vacuum to obtain the second modified amino resin. Dissolve 2.5 g of Rhizomucor miehei lipase in 50 mL of phosphate buffer (pH 7, 50 mM), centrifuge and take the supernatant to obtain the lipase solution. Take 2 mL of the above lipase solution, add 0.2 g of the second modified amino resin, immobilize at 35 °C for 8 h respectively, filter to separate the immobilized lipase, wash the immobilized lipase with phosphate buffer until there is no protein residue in the filtrate, and vacuum dry the washed immobilized product at 40 °C for 4 h.

[0064] The properties of the obtained immobilized lipase were characterized, and the results showed that the immobilization efficiency was 65.52% and the enzyme activity was 9769.58 U / g. Comparative Example 1

[0065] Take 4 mL of the lipase solution in Example 1, add 0.2 g of LX-1000HAA amino resin after vacuum drying, immobilize at 30 °C for 10 h, filter and separate the immobilized lipase, wash the immobilized lipase with phosphate buffer until there is no protein residue in the filtrate, and vacuum dry the washed immobilized product at 40 °C for 4 h.

[0066] The properties of the obtained immobilized lipase were characterized, and the results showed that the immobilization efficiency was 31.91% and the enzyme activity was 5995.66 U / g. Comparative Example 2

[0067] Weigh 10 g of wet LX-1000HAA resin into 200 mL of sodium bicarbonate buffer (pH 8.5, 50 mM), add 0.4 g of tannic acid (the concentration of tannic acid dissolved in buffer A is 2 mg / mL), stir at room temperature for 12 h, filter and vacuum dry to obtain the first modified amino resin. Take 4 mL of the lipase solution in Example 1, add 0.2 g of the first modified amino resin, immobilize at 30 °C for 10 h, filter and separate the immobilized lipase, wash the immobilized lipase with phosphate buffer until there is no protein residue in the filtrate, and vacuum dry the washed immobilized product at 40 °C for 4 h.

[0068] The properties of the obtained immobilized lipase were characterized, and the results showed that the immobilization efficiency was 35.14% and the enzyme activity was 6895.43 U / g.

[0069] The comparison of the characterization effects of Examples 1-11 and Comparative Examples 1 and 2 is shown in Table 3. Compared with the amino resin, the enzyme activity and immobilization efficiency of the immobilized lipase using the modified amino resin as the carrier were significantly improved. Under the optimal test conditions, the enzyme activity was increased by up to 94.12% compared with the amino resin.

[0070]

[0071] Take the immobilized lipase of the second modified amino resin grafted with octadecylamine in Example 1 and the immobilized lipase of the amino resin in Comparative Example 1, and incubate them at 40 °C and 60 °C for 10 h respectively, and regularly sample to measure the enzyme activity. The results are as Figure 7As shown in the figure, at 40 °C and 60 °C environments, the decline in the activity of the amino resin-immobilized lipase was higher than that of the second-modified amino resin-immobilized lipase. Among them, when incubated at 60 °C for 10 h, the second-modified amino resin-immobilized lipase still retained 79.15% of its initial enzyme activity, while the amino resin-immobilized lipase was only 57.92%. The results show that the thermal stability of the second-modified amino resin-immobilized lipase was significantly improved compared with that of the amino resin-immobilized lipase.

[0072] Take the second-modified amino resin-immobilized lipase grafted with octadecylamine in Example 1 and the amino resin-immobilized lipase in Comparative Example 1, and store them at 4 °C environment respectively. Samples were taken regularly to measure the enzyme activity. The results are as Figure 8 shown. At 4 °C environment, the decline in the activity of the amino resin-immobilized lipase was significantly higher than that of the second-modified amino resin-immobilized lipase. Among them, after storing at 4 °C for 30 days, the second-modified amino resin-immobilized lipase still retained 95.35% of its initial enzyme activity, while the amino resin-immobilized lipase was only 80.13%. The results show that the storage stability of the second-modified amino resin-immobilized lipase was significantly improved compared with that of the amino resin-immobilized lipase.

[0073] In the examples and comparative examples of the present invention, the method for detecting protein residues in the filtrate was detected by Coomassie Brilliant Blue method; other treatment methods not described in detail, such as filtration, vacuum drying, etc., can be processed according to the conventional treatment methods in this industry, and will not be elaborated in this application.

[0074] The above is only the preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A method for preparing immobilized lipase using a graft-modified amino resin as a carrier, characterized in that, It includes the following steps: Step S1: Dissolve tannic acid in an alkaline buffer solution A, add amino resin, carry out an oxidation reaction at room temperature, and obtain a first modified amino resin after filtration and drying; Step S2: Add fatty amine and the first modified amino resin into an ethanol solution respectively, carry out a grafting reaction, and obtain a second modified amino resin after filtration and drying; Step S3: Add the second modified amino resin into a lipase solution, filter out the liquid after adsorption, wash it with buffer solution B, and obtain immobilized lipase after drying.

2. The method for preparing immobilized lipase using a grafted modified amino resin as a carrier according to claim 1, characterized in that: In step S1, the concentration of tannic acid dissolved in buffer solution A is 1 mg / mL - 5 mg / mL.

3. A method for preparing immobilized lipase using a graft-modified amino resin as a carrier according to claim 2, characterized in that, In step S1, buffer solution A is sodium bicarbonate buffer solution or Tris - hydrochloric acid buffer solution or HEPES buffer solution, and the pH of buffer solution A is 8 - 9.

4. A method for preparing immobilized lipase using a graft-modified amino resin as a carrier according to claim 1, characterized in that, In step S1, the model of the amino resin is one of LX - 1000HAA, LX - 1000EPN, LX - 1000EPHA, LX - 1000NH, and LX - 1000EA.

5. A method for preparing immobilized lipase using a graft-modified amino resin as a carrier according to claim 1, characterized in that, In step S2, the fatty amine includes one or more of hexylamine, octylamine, decylamine, dodecylamine, tetradecylamine, hexadecylamine, and octadecylamine.

6. A method for preparing immobilized lipase using a graft-modified amino resin as a carrier according to claim 5, characterized in that, In step S2, the addition amount of the fatty amine is 0.2 mmol - 1 mmol; the concentration of the ethanol solution is 50% - 100%; the grafting reaction temperature is 40 °C - 60 °C; the grafting reaction time is 4 - 8 h.

7. A method for preparing immobilized lipase using a graft-modified amino resin as a carrier according to claim 1, characterized in that, In step S3, the mass - volume ratio of the second modified amino resin to the lipase solution is 1 g∶5 - 25 mL.

8. A method for preparing immobilized lipase using a graft-modified amino resin as a carrier according to claim 6, characterized in that, In step S3, the pH of the lipase solution is 5 - 10, the adsorption time is 2 - 10 h, and the adsorption temperature is 25 - 45 °C.

9. A method for preparing immobilized lipase using a graft-modified amino resin as a carrier according to claim 1, characterized in that, The lipase is Rhizomucor miehei lipase.

10. An immobilized lipase prepared using a grafted modified amino resin as a carrier, characterized in that: It is prepared by the method according to any one of claims 1 - 9.

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